Lyme disease is the most common vector-borne disease in the United States. It is caused by the bite of a tick infected with the spirochete Borrelia burgdorferi, the etiologic agent of the disease. Current laboratory diagnosis rests on a number of serologic tests of varying degrees of sensitivity and reliability. This limits the rapid and specific diagnosis of the disease immediately following a tick bite. Furthermore, evaluation of the potential for development of disease from a tick bite is complicated by incomplete knowledge of the diversity of spirochete genotypes in nature and their pathogenic capacity. In order to address these public health risks the following studies will be pursued: 1) An rRNA-based assay for the direct detection of Borrelia burgdorferi will be further refined. The assay will be designed around a streptavidin-coated microwell plate format using biotinylated capture probes and enzyme-linked detection probes. This assay will obviate the requirement for PCR amplification, making it simpler and more rapid. 2) A molecular typing assay for the genotypic analysis of B. burgdorferi which can be used without the need for prior culture was developed. This assay will be employed to analyze the diversity of B. burgdorferi species in ticks from different locations in the Northeastern United States, in vertebrate reservoir hosts and in samples obtained from patients with clinically confirmed Lyme disease. 3) The PCR-based assay for detection of B. burgdorferi developed during the currently funded project will be employed in further studies on the distribution of the spirochete in nature and in Lyme disease patients with the objective of increasing the diagnostic value of the assay in clinical specimens. The proposed studies should result in a simpler and more rapid method for detection of B. burgdorferi which, in turn, will have many applications in Lyme disease research. They will also provide a more complete understanding of the molecular diversity of B. burgdorferi species in nature and yield insights into the potential relationship between spirochete genotypes and clinical disease.